Cypher/ZASP drives cardiomyocyte maturation via actin-mediated MRTFA-SRF signalling

Rationale: Cardiomyocytes (CMs) undergo dramatic structural and functional changes in postnatal maturation; however, the regulatory mechanisms remain greatly unclear. Cypher/Z-band alternatively spliced PDZ-motif protein (ZASP) is an essential sarcomere component maintaining Z-disc stability. Deletion of mouse Cypher and mutation in human ZASP result in dilated cardiomyopathy (DCM). Whether Cypher/ZASP participates in CM maturation and thereby affects cardiac function has not been answered. Methods: Immunofluorescence, transmission electron microscopy, real-time quantitative PCR, and Western blot were utilized to identify the role of Cypher in CM maturation. Subsequently, RNA sequencing and bioinformatics analysis predicted serum response factor (SRF) as the key regulator. Rescue experiments were conducted using adenovirus or adeno-associated viruses encoding SRF, both in vitro and in vivo. The molecular mechanisms were elucidated through G-actin/F-actin fractionation, nuclear-cytoplasmic extraction, actin disassembly assays, and co-sedimentation assays. Results: Cypher deletion led to impaired sarcomere isoform switch and morphological abnormalities in mitochondria, transverse-tubules, and intercalated discs. RNA-sequencing analysis revealed significant dysregulation of crucial genes related to sarcomere assembly, mitochondrial metabolism, and electrophysiology in the absence of Cypher. Furthermore, SRF was predicted as key transcription factor mediating the transcriptional differences. Subsequent rescue experiments showed that SRF re-expression during the critical postnatal period effectively rectified CM maturation defects and notably improved cardiac function in Cypher-depleted mice. Mechanistically, Cypher deficiency resulted in the destabilization of F-actin and a notable increase in G-actin levels, thereby impeding the nuclear localisation of myocardin-related transcription factor A (MRTFA) and subsequently initiating SRF transcription. Conclusion: Cypher/ZASP plays a crucial role in CM maturation through actin-mediated MRTFA-SRF signalling. The linkage between CM maturation abnormalities and the late-onset of DCM is suggested, providing further insights into the pathogenesis of DCM and potential treatment strategies.


Figure S2 .
Figure S2.Ultrastructural disorders related to CM maturation in the progression of dilated cardiomyopathy caused by Cypher deletion.Electron micrographs showing the ultrastructural characteristics of CMs in WT and CypherL-KO mice at the age of 2 months (A, B), 6 months (C, E) and 12 months (D, F).Sarcomere length and width are highlighted using blue or red lines.T-tubules and intercalated discs are indicated by red and green arrows, respectively.Scale bars: 2 or 1 μm.Quantitative analysis (n = 3).Data represent the mean ± SEM.Unpaired two-tailed Student's t test (B, E, F), ****p < 0.0001.

Figure S3 .
Figure S3.Effect of SRF re-expression on the maturation of cardiomyocytes and the cardiac function of mice.(A-B) Western blot analysis of SRF, immature (Myh7 and Tnni1), and mature (Myh6 and Tnni3) myofibrillar isoforms in neonatal NRCMs infected with control adenovirus (Ad-GFP) or adenovirus carrying SRF (Ad-SRF), GAPDH was probed as a loading control.Statistical results in (B, n = 3).Similar results were observed in 3 independent experiments.(C-D) The protein levels of SRF in WT and CypherL-KO hearts at 21 days of age after adeno-associated virus encoding SRF (AAV-SRF) or control AAV-EGFP injection.GAPDH was used as the internal reference.Quantifications are shown in D (n = 3).(E) WGA staining in cardiac tissue sections from P21 WT and CypherL-KO mice after AAV-SRF or AAV-EGFP injection.DAPI was used to label nuclei.Scale bars: 50 μm.(F) Statistical results of CM diameters (n = 3).(G) Quantification of length, width, length-to-width ratio, and cell area of CMs in P21 WT and CypherL-KO mice infected with AAV-SRF or AAV-EGFP (n = 3).(H) RT-qPCR analysis of cardiac foetal genes (Nppa and Nppb) in P21 WT and CypherL-KO mice infected with AAV-SRF or control AAV-EGFP (n = 6), with GAPDH as a reference gene.(I) Electron micrographs of sarcomere structures in P21 WT and CypherL-KO hearts injected with AAV-SRF or control AAV-EGFP.Sarcomere length and width are highlighted using blue or red lines.Scale bars: 1 μm.(J-K) Statistical analysis (n = 3).(L) WGA and actinin co-staining of cardiac tissue sections from P21 WT and CypherL-KO mice injected with AAV-SRF or control AAV-EGFP.Scale bars: 25 μm.(M) Quantitative analysis of sarcomere numbers within each cardiomyocyte (n = 3).(N-O) Cardiac function in 2-and 6-month-old WT and CypherL-KO mice injected with AAV-EGFP or AAV-SRF.Echocardiographic parameters in 2-month-old mice (N, n = 7-8) and 6-month-old mice (O, n = 7-8).EF, ejection fraction; FS, fractional shortening; LVIDs, left ventricle internal dimension at systole; LVPWs, left ventricular posterior wall thickness at systole; ESV, end-systolic volume.Data represent the mean ± SEM.Unpaired two-tailed Student's t test (B) and two-way ANOVA followed by Bonferroni's post hoc test (D-O), *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significant difference.

Figure S4 .
Figure S4.The re-expression of SRF from postnatal one month did not exhibit any ameliorative effect on cardiac function in CypherL-KO mice.(A) Schematic diagram of the experimental strategy.One-month-old WT and CypherL-KO mice were injected subcutaneously with AAV-SRF or control AAV-EGFP, and sacrificed after echocardiography detection at the age of 6 months.(B-C) The protein level of SRF in WT and CypherL-KO hearts at 21 days after AAV-EGFP or AAV-SRF injection.GAPDH was used as the internal reference (n = 3).(D) Representative H&E images of cardiac tissue sections from 6-month-old WT and CypherL-KO mice infected with AAV-SRF or control AAV-EGFP.Scale bars: 2 mm.(E) Representative echocardiographs of 6-month-old WT and CypherL-KO mice injected with AAV-SRF or control AAV-EGFP.(F) Statistical analysis of echocardiographic parameters.EF, ejection fraction; FS, fractional shortening; LVIDs, left ventricle internal dimension at systole; LVPWs, left ventricular posterior wall thickness at systole; ESV, end-systolic volume.(n = 7).Data represent the mean ± SEM.Two-way ANOVA followed by Bonferroni's post hoc test (C, F), *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significant difference.

Figure S5 .
Figure S5.Cypher modulates the SRF-MRTFA pathway without direct interaction or affecting Rho GTPase activity.(A-B) Western blots of SRF and MRTFA in NRCMs treated with SiScramble, SiCypher, and SiCypher upon Ad-SRF infection, with GAPDH as a loading control.Quantitative analysis (n = 3).(C-D) Coimmunoprecipitation of Cypher and SRF or MRTFA in 293T cells.Myc-tagged Cypher and Flag-tagged MRTFA (C) or SRF (D) were co-transfected to 293T cells.Immunoprecipitation was performed using anti-Flag (C) or anti-Myc (D) antibody, and the samples were further probed with anti-Myc or anti-Flag antibody.IP, immunoprecipitation; IB, immunoblotting.Mouse IgG served as the negative control.(E-F) Protein levels of Rho GTPases (Rhoa, Rac1, Cdc42) and their active GTP-bound forms in NRCMs treated with SiScramble or SiCypher, GAPDH was used as a loading control.Statistical results in F (n = 3).Similar results were obtained from 3 independent cellular experiments.Data represent the mean ± SEM.Unpaired two-tailed Student's t test (F) and one-way ANOVA with Tukey's post hoc test (B), *p < 0.05; ***p < 0.001; ****p < 0.0001; ns, no significant difference.

Figure S7 .
Figure S7.Cypher deficiency disturbs actin dynamics and MRTFA nuclear localisation.(A-B) Immunofluorescence images of SiScramble-or SiCyphertransfected NRCMs after latrunculin B washout.NRCMs were treated with 10 μM latrunculin B for 1 h, followed by 2 or 4 h drug washout.F-actin and cardiomyocytes were labelled with phalloidin and α-actinin, respectively.Scale bars: 25 μm.F-actin fluorescence intensity (B), 6 randomly selected fields of view were quantified for each group.(C-D) Immunostaining for MRTFA in NRCMs after latrunculin B washout.Nuclei were labelled DAPI.Scale bars: 25 μm.Nuclear to cytoplasmic MRTFA ratio (D), 6 randomly selected fields of view were quantified for each group.Data represent the mean ± SEM.Similar results were obtained from 3 independent cellular experiments.Two-way ANOVA followed by Bonferroni's post hoc test (B and D), ***p < 0.001; ****p < 0.0001; ns, no significant difference.